نحن ممتنون للغاية لجميع أعضاء مختبر ماو وهيو ، في الماضي والحاضر ، على المناقشات الشيقة والمساهمات العظيمة في المشروع. نحن ممتنون للمركز الوطني للبحوث السريرية لصحة الطفل على الدعم الكبير. تم دعم هذه الدراسة ماليا من قبل المؤسسة الوطنية للعلوم الطبيعية في الصين (U20A20351 إلى جيانهوا ماو ، 82200784 إلى ليدان هو) ، ومؤسسة العلوم الطبيعية في مقاطعة تشجيانغ الصينية (No. LQ22C070004 إلى ليدان هو).

توصيف المستضدات السطحية والقدرة على الانتشار في MSCs التي تقود hiPSC

<ol>
	<li>Weng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem/stromal+cell+senescence:+Hallmarks,+mechanisms,+and+combating+strategies.">Mesenchymal stem/stromal cell senescence: Hallmarks, mechanisms, and combating strategies.</a> Stem Cells Translational Medicine. 11 (4), 356-371 (2022).</li><li>Soliman, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stromal+cells:+One+name,+multiple+identities.">Multipotent stromal cells: One name, multiple identities.</a> Cell Stem Cell. 28 (10), 1690-1707 (2021).</li><li>Friedenstein, A. J., Chailakhyan, R. K., Gerasimov, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+osteogenic+stem+cells:+in+vitro+cultivation+and+transplantation+in+diffusion+chambers.">Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers.</a> Cell and Tissue Kinetics. 20 (3), 263-272 (1987).</li><li>Friedenstein, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursors+for+fibroblasts+in+different+populations+of+hematopoietic+cells+as+detected+by+the+in+vitro+colony+assay+method.">Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.</a> Experimental Hematology. 2 (2), 83-92 (1974).</li><li>Friedenstein, A. J., Gorskaja, J. F., Kulagina, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+precursors+in+normal+and+irradiated+mouse+hematopoietic+organs.">Fibroblast precursors in normal and irradiated mouse hematopoietic organs.</a> Experimental Hematology. 4 (5), 267-274 (1976).</li><li>El Agha, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+fibrotic+disease.">Mesenchymal stem cells in fibrotic disease.</a> Cell Stem Cell. 21 (2), 166-177 (2017).</li><li>Mushahary, D., Spittler, A., Kasper, C., Weber, V., Charwat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation,+and+characterization+of+human+mesenchymal+stem+cells.">Isolation, cultivation, and characterization of human mesenchymal stem cells.</a> Cytometry A. 93 (1), 19-31 (2018).</li><li>Ullah, M., Liu, D. D., Thakor, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell+homing:+Mechanisms+and+strategies+for+improvement.">Mesenchymal stromal cell homing: Mechanisms and strategies for improvement.</a> iScience. 15, 421-438 (2019).</li><li>Regmi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+viability+and+function+of+mesenchymal+stromal+cell+spheroids+is+mediated+via+autophagy+induction.">Enhanced viability and function of mesenchymal stromal cell spheroids is mediated via autophagy induction.</a> Autophagy. 17 (10), 2991-3010 (2021).</li><li>Hoang, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+therapy+for+human+diseases.">Stem cell-based therapy for human diseases.</a> Signal Transduction and Targeted Therapy. 7 (1), 272 (2022).</li><li>Jiang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Mesenchymal+stem+cells+derived+from+human+pluripotent+cells,+an+unlimited+and+quality-controllable+source+for+therapeutic+applications.">Concise review: Mesenchymal stem cells derived from human pluripotent cells, an unlimited and quality-controllable source for therapeutic applications.</a> Stem Cells. 37 (5), 572-581 (2019).</li><li>Soontararak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+(MSC)+derived+from+induced+pluripotent+stem+cells+(iPSC)+equivalent+to+adipose-derived+MSC+in+promoting+intestinal+healing+and+microbiome+normalization+in+mouse+inflammatory+bowel+disease+model.">Mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) equivalent to adipose-derived MSC in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model.</a> Stem Cells Translational Medicine. 7 (6), 456-467 (2018).</li><li>Di Baldassarre, A., Cimetta, E., Bollini, S., Gaggi, G., Ghinassi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cell+technology+and+cardiomyocyte+generation:+Progress+and+clinical+applications.">Human-induced pluripotent stem cell technology and cardiomyocyte generation: Progress and clinical applications.</a> Cells. 7 (6), 48 (2018).</li><li>Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+technology:+a+decade+of+progress.">Induced pluripotent stem cell technology: a decade of progress.</a> Nature Reviews. Drug Discovery. 16 (2), 115-130 (2017).</li><li>Levy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shattering+barriers+toward+clinically+meaningful+MSC+therapies.">Shattering barriers toward clinically meaningful MSC therapies.</a> Science Advances. 6 (30), eaba6884 (2020).</li><li>Zhao, C., Ikeya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+applications+of+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells.">Generation and applications of induced pluripotent stem cell-derived mesenchymal stem cells.</a> Stem Cells International. 2018, 9601623 (2018).</li><li>Path, G., Perakakis, N., Mantzoros, C. S., Seufert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+treatment+of+diabetes+mellitus+-+Focus+on+mesenchymal+stem+cells.">Stem cells in the treatment of diabetes mellitus - Focus on mesenchymal stem cells.</a> Metabolism. 90, 1-15 (2019).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+derivation+of+functional+mesenchymal+stem+cells+from+human+pluripotent+stem+cells.">One-step derivation of functional mesenchymal stem cells from human pluripotent stem cells.</a> Bio-Protocol. 8 (22), e3080 (2018).</li><li>Hua, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-intensity+pulsed+ultrasound+promotes+osteogenic+potential+of+iPSC-derived+MSCs+but+fails+to+simplify+the+iPSC-EB-MSC+differentiation+process.">Low-intensity pulsed ultrasound promotes osteogenic potential of iPSC-derived MSCs but fails to simplify the iPSC-EB-MSC differentiation process.</a> Frontiers in Bioengineering and Biotechnology. 10, 841778 (2022).</li><li>Dupuis, V., Oltra, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+produce+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells:+Mesenchymal+stem+cells+from+induced+pluripotent+stem+cells.">Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells.</a> World Journal of Stem Cells. 13 (8), 1094-1111 (2021).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+stem+cells.+A+Werner+syndrome+stem+cell+model+unveils+heterochromatin+alterations+as+a+driver+of+human+aging.">Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.</a> Science. 348 (6239), 1160-1163 (2015).</li><li>Liu, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+Fanconi+anemia+pathogenesis+and+therapeutics+using+integration-free+patient-derived+iPSCs.">Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.</a> Nature Communications. 5, 4330 (2014).</li><li>Kubben, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+the+antioxidant+NRF2+pathway+in+premature+aging.">Repression of the antioxidant NRF2 pathway in premature aging.</a> Cell. 165 (6), 1361-1374 (2016).</li><li>Duan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+deficiency+reprogrammes+human+neural+stem+cells+towards+a+glioblastoma+stem+cell-like+phenotype.">PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype.</a> Nature Communications. 6, 10068 (2015).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+released+from+human+induced+pluripotent+stem+cells-derived+MSCs+facilitate+cutaneous+wound+healing+by+promoting+collagen+synthesis+and+angiogenesis.">Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis.</a> Journal of Translational Medicine. 13, 49 (2015).</li><li>Hu, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+secreted+by+human-induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells+attenuate+limb+ischemia+by+promoting+angiogenesis+in+mice.">Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.</a> Stem Cell Research &amp; Therapy. 6 (1), 10 (2015).</li><li>Kang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+human+induced+pluripotent+stem+cells+retain+adequate+osteogenicity+and+chondrogenicity+but+less+adipogenicity.">Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.</a> Stem Cell Research &amp; Therapy. 6 (1), 144 (2015).</li><li>Wang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mesenchymal+stem+cells+from+human+induced+pluripotent+stem+cells+results+in+downregulation+of+c-Myc+and+DNA+replication+pathways+with+immunomodulation+toward+CD4+and+CD8+cells.">Differentiation of mesenchymal stem cells from human induced pluripotent stem cells results in downregulation of c-Myc and DNA replication pathways with immunomodulation toward CD4 and CD8 cells.</a> Stem Cells. 36 (6), 903-914 (2018).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Kim, S., Kim, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem-like+cells+for+producing+extracellular+vesicles.">Generation of mesenchymal stem-like cells for producing extracellular vesicles.</a> World Journal of Stem Cells. 11 (5), 270-280 (2019).</li></ol>

ليس لدى المؤلفين ما يكشفون عنه.

في هذا البروتوكول ، تم فحص طريقتين تمثيليتين للتمييز بين hiPSCs إلى MSCs20،21،22،23،24،25،26،27،28،30. كانت كلتا الط?...

الخلايا اللحمية المتوسطة (MSCs) هي خلايا جذعية بالغة متعددة القدرات مشتقة من الأديم المتوسط1. MSCs موجودة في جميع الأنسجة الضامة تقريبا2. منذ اكتشاف MSCs لأول مرة في سبعينيات القرن العشرين وعزلها بنجاح من نخاع العظام في عام 1987 من قبل Friedenstein et al.3،4،5 ، تم استخدام مجموعة متنوعة من الأنسجة الجسدية البشرية (بما في ذلك الجنين والبالغين) لعزل MSCs مثل العظام والغضاريف والأوتار والعضلات والأنسجة الدهنية ، والسدى الداعم للدم1،2،6،7. تظهر MSCs قدرات تكاثرية عالية ومرونة للتمايز إلى العديد من سلالات الخلايا الجسدية ويمكن أن تهاجر إلى الأنسجة المصابة والملتهبة2،8،9. هذه الخصائص تجعل MSCs مرشحا محتملا للطب التجديدي10. ومع ذلك ، فإن MSCs المشتقة من الأنسجة الجسدية (st-MSCs) مقيدة بالتبرع المحدود ، والقدرة المحدودة على تكاثر الخلايا ، واختلافات الجودة ، ومخاوف السلامة البيولوجية للانتقال المحتمل لمسببات الأمراض ، إن وجدت ، من المتبرعين11،12.
تستمد الخلايا الجذعية متعددة القدرات التي يسببها الإنسان (hiPSCs) من إعادة برمجة الخلايا البالغة بعوامل النسخ (Oct4 و Sox2 و Klf4 و c-Myc) ، والتي لها وظائف مماثلة للخلايا الجذعية الجنينية13,14. يمكنهم التجديد الذاتي وامتلاك إمكانية التمايز إلى أي نوع من الخلايا الجسدية ، بما في ذلك MSCs. بالمقارنة مع st-MSCs ، تتمتع iPSC-MSCs بميزة العرض غير المحدود ، والتكلفة المنخفضة ، والنقاء العالي ، والراحة في مراقبة الجودة ، وسهولة الإنتاج على نطاق واسع وتعديل الجينات15،16،17.
نظرا لهذه المزايا ل iPSC-MSCs ، تم الإبلاغ عن مجموعة متنوعة من الطرق التي تقود MSC من iPSC. تركزت طرق التمايز هذه حول منهجيتين للاستزراع: (1) تكوين الأجسام الجنينية (EBs) و (2) استخدام الثقافات أحادية الطبقة11،18،19،20. وهنا تم تحديد نهج تمثيلي لكل من المنهجيتين. علاوة على ذلك ، تم أيضا الوصول إلى مقارنات بين نهجين تمثيليين على أساس الوقت والتكلفة والقدرة على التكاثر والتعبير عن المؤشرات الحيوية MSC والقدرة على التمايز في المختبر .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alizarin red staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0148S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD105 (PE)</td>
			<td>Biolegend</td>
			<td>323206</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD34 (FITC)</td>
			<td>Biolegend</td>
			<td>343503</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD45 (APC)</td>
			<td>Biolegend</td>
			<td>304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD73( APC)</td>
			<td>Biolegend</td>
			<td>344006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD90 (FITC)</td>
			<td>Biolegend</td>
			<td>328108</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Solarbio</td>
			<td>A8100</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-6</td>
			<td>Novoprotein</td>
			<td>C012</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide level shaker</td>
			<td>Crystal</td>
			<td>CO-06UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation Beads</td>
			<td>BioLegend</td>
			<td>424601</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>STEMCELL Technology</td>
			<td>07959</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160; medium</td>
			<td>Servicebio</td>
			<td>G4610</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HAKATA</td>
			<td>HS-FBS-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Stemcell</td>
			<td>78003.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control APC</td>
			<td>BioLegend</td>
			<td>403505</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control FITC</td>
			<td>BioLegend</td>
			<td>403507</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control PE</td>
			<td>BioLegend</td>
			<td>403503</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#946;1</td>
			<td>Stemcell</td>
			<td>78067</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX&#160;</td>
			<td>BioLegend</td>
			<td>422301</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Solarbio</td>
			<td>SI9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>P3376</td>
			<td></td>
		</tr>
		<tr>
			<td>iPSC maintenance medium</td>
			<td>STEMCELL Technology</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Media Supplement</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0341-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced</td>
			<td>BD Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Oli Red O staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0158S</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Solarbio</td>
			<td>P0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;3</td>
			<td>Novoprotein</td>
			<td>CJ44</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue staining kit</td>
			<td>Solarbio</td>
			<td>G2543</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme(1x)&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 6 Well Plate</td>
			<td>Costar</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stemcell</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Hyclone</td>
			<td>SH30265</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Solarbio</td>
			<td>G8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparison of two representative methods for differentiation of human induced pluripotent stem cells into mesenchymal stromal cells

الخلايا اللحمية المتوسطة (MSCs) هي خلايا جذعية بالغة متعددة القدرات تستخدم على نطاق واسع في الطب التجديدي. نظرا لأن MSCs المشتقة من الأنسجة الجسدية مقيدة بالتبرع المحدود واختلافات الجودة والسلامة البيولوجية ، فقد شهدت السنوات ال 10 الماضية ارتفاعا كبيرا في الجهود المبذولة لتوليد MSCs من الخلايا الجذعية متعددة القدرات التي يسببها الإنسان (hiPSCs). تركزت الجهود السابقة والحديثة في تمايز hiPSCs إلى MSCs حول منهجيتين للاستزراع: (1) تكوين الأجسام الجنينية (EBs) و (2) استخدام الثقافة أحادية الطبقة. يصف هذا البروتوكول هاتين الطريقتين التمثيليتين في اشتقاق MSC من hiPSCs. تقدم كل طريقة مزاياها وعيوبها ، بما في ذلك الوقت والتكلفة والقدرة على تكاثر الخلايا والتعبير عن علامات MSC وقدرتها على التمايز في المختبر. يوضح هذا البروتوكول أن كلتا الطريقتين يمكن أن تشتق MSCs ناضجة ووظيفية من hiPSCs. تتميز طريقة الطبقة الأحادية بتكلفة أقل ، وتشغيل أبسط ، وتمايز عظمي أسهل ، بينما تتميز طريقة EB باستهلاك أقل للوقت.

Mesenchymal stromal cells (MSCs) are mesoderm-derived adult pluripotent stem cells1. MSCs are present in almost all connective tissues2. Since MSCs were first discovered in the 1970s and successfully isolated from bone marrow in 1987 by Friedenstein et al.3,4,5, a variety of human somatic (including fetal and adult) tissues have been used for isolating MSCs such as bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic-supporting stroma1,2,6,7. MSCs demonstrate high proliferative capabilities and plasticity to differentiate into many somatic cell lineages and could migrate to injured and inflamed tissues2,8,9. These properties make MSCs a potential candidate for regenerative medicine10. However, somatic tissue-derived MSCs (st-MSCs) are restricted by limited donation, limited cell proliferative capacity, quality variations, and biosafety concern for possible transmission of pathogens, if any, from the donors11,12.
Human induced pluripotent stem cells (hiPSCs) are derived from adult cells reprogramming with transcription factors (Oct4, Sox2, Klf4, and c-Myc), which have similar functions as embryonic stem cells13,14. They can self-renew and possess the potential of differentiating into any type of somatic cells, including MSCs. Compared with st-MSCs, iPSC-MSCs has the advantage of unlimited supply, lower cost, higher purity, convenience in quality control, easy for scale production and gene modification15,16,17.
Due to these advantages of iPSC-MSCs, a variety of methods driving MSC from iPSC have been reported. These differentiation methods have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer cultures11,18,19,20. Herein, a representative approach for each of the two methodologies was characterized. Furthermore, comparisons between two representative approaches based on time, cost, proliferative ability, expression of MSC biomarkers, and differentiation capability in vitro were also accessed.

تسليط الضوء على المؤلف: التطورات في iPSCs وأبحاث الأمراض الوراثية 

مقارنة بين طريقتين تمثيليتين لتمايز الخلايا الجذعية متعددة القدرات المستحثة بشريا إلى خلايا لحمية وسيطة

The scope of our study is a mechanism, and a therapeutic strategy research of genetic disease. Our research is based on multilevel disease models, such as cell lines, iPSCs, organoids, and mice. iPSCs have a distinct advantage over other models.iPSCs from patients, and any subsequent derived cells and organoids share the same genome with the patients, which provides a bank of patient cells and models for many disease. Also, with the methods of established iPSCs from patients are quite mature. Many challenges remain in the direction of differentiating of iPSCs into specific cells and organoids, with a deepening understanding of development mechanisms in current development will occur in the field.

باتباع البروتوكول (الشكل 1 أ) ، تم تمييز hiPSCs إلى MSCs عبر تكوين EB وطرق الاستزراع أحادي الطبقة. أثناء التمايز ، أظهرت الخلايا مورفولوجيات تمثيلية مختلفة (الشكل 1B ، C).
كما هو موضح في الشكل 1B ، تعرض مستعمرات hiPSCs مورفولوجيا م...

Watch this Scientific Journal Video about Advancements in iPSCs and Genetic Disease Research at JoVE.com

يصف هذا البروتوكول ويقارن طريقتين تمثيليتين للتمييز بين hiPSCs إلى خلايا انسجة اللحمة المتوسطة (MSCs). تتميز طريقة الطبقة الأحادية بتكلفة أقل وتشغيل أبسط وتمايز عظمي أسهل. تتميز طريقة الأجسام الجنينية (EBs) باستهلاك أقل للوقت.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

نطاق دراستنا هو آلية ، واستراتيجية علاجية للبحث عن الأمراض الوراثية. يعتمد بحثنا على نماذج الأمراض متعددة المستويات ، مثل خطوط الخلايا ، و iPSCs ، والمواد العضوية ، والفئران. تتمتع iPSCs بميزة واضحة مقارنة بالموديلات الأخرى.iPSCs من المرضى ، وأي خلايا مشتقة لاحقة وعضويات تشترك في نفس الجينوم مع المرضى ، مما يوفر بنكا لخلايا المريض ونماذج للعديد من الأمراض. أيضا ، مع أساليب iPSCs المعمول بها من المرضى ناضجة جدا. لا تزال هناك العديد من التحديات في اتجاه التمييز بين iPSCs إلى خلايا وعضويات محددة ، مع تعميق فهم آليات التطوير في التطوير الحالي سيحدث في هذا المجال.

comparison-two-representative-methods-for-differentiation-human

Research

JoVE Journal

Biology

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

773 Views.  The Children's Hospital, Zhejiang University School of Medicine. نطاق دراستنا هو آلية ، واستراتيجية علاجية للبحث عن الأمراض الوراثية. يعتمد بحثنا على نماذج الأمراض متعددة المستويات ، مثل خطوط الخلايا ، و iPSCs ، والمواد العضوية ، والفئران. تتمتع iPSCs بميزة واضحة مقارنة بالموديلات الأخرى.iPSCs من المرضى ، وأي خلايا مشتقة لاحقة وعضويات تشتر?...

Video: تسليط الضوء على المؤلف: التطورات في iPSCs وأبحاث الأمراض الوراثية 

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

MSCs Differentiation from hiPSCs via EB Formation and Monolayer Culture

To begin, culture hiPSCs in IPSC maintenance medium on a six-well tissue culture plate coated with growth factor reduced extracellular matrix gel. When the cells attain 80 to 90%v, remove the medium from the plate and wash the cells once with DPBS. Then add 700 to 800 microliters of 0.48 millimolar EDTA and incubate for one minute at room temperature.Remove the digestion solution and incubate the plate at 37 degrees Celsius for three to five minutes. When cells are digested into sheets, add two milliliters of IPSCs maintenance medium to terminate digestion. Transfer the cell suspension to a six-well low attachment plate.Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 60 RPM to form spherical embryo bodies or EBs. After 24 hours, using a past pipette, transfer the EBs to the centrifuge tube and allow it to sediment for five to 10 minutes before removing the supernatant. Add MSC differentiation medium to the tube and transfer the EBs to a six-well low attachment blade with two milliliters of MSCs differentiation medium.Culture the EBs on the shaker at 37 degrees Celsius with 5%carbon dioxide for seven days. On the eighth day, transfer the EBs to the centrifuge tube as demonstrated previously. Once the EBs are sedimented, remove the supernatant from the tube.Add MSC maintenance medium into the tube and transfer the EBs to a growth factor reduced extracellular matrix gel coated six-well plate with two milliliters of MSC maintenance medium. After cell adhesion, change the medium until the culture attains 90%confluency. Next, treat the EB derived culture with a dissociation solution at 37 degrees Celsius.When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium to terminate digestion and transfer the cell suspension to a centrifuge tube. Wash the remaining undigested cells from the wells once with DPBS. And digest again as demonstrated previously.Then centrifuge the cell suspension at 250g for five minutes. Remove the supernatant and seed the cells in a gelatin coated culture plate. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.Culture hiIPCs lines as demonstrated previously. When the cells attain 50 to 60%confluency, remove the IPSC maintenance medium from the plate. Add two milliliters of MSC maintenance medium.And culture for 14 days at 37 degrees Celsius and 5%carbon dioxide with daily medium change. For the maturation of MSCs, treat the monolayer culture with a dissociation solution at 37 degrees Celsius. When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium into the wells to terminate digestion and transfer the cell suspension to a centrifuge tube.Wash the remaining undigested cells once with DPBS and digest them as demonstrated previously. Then centrifuge the cell suspension as demonstrated previously and seed the cells on a gelatin coated culture dish. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.In the EB formation method, hiIPCs colonies exhibited a compact morphology before differentiation. After dissociation, uniform spherical EBs formed, which drew in volume over time. Subsequently, EBs transformed into adherent monolayer cells achieving 90%confluency by day 18 and acquiring a spindle shape morphology after two passages.Similarly, in the monolayer method, cells proliferated forming multilayer adherent cells. After packaging multiple times, cells matured into MSC with a typical spindle shape and swirling colony. Flow cytometry analysis revealed that hiIPCs were positive for CD90 and negative for CD34, CD45, CD105, and CD73.After differentiation, the hiIPCs driving MSCs expressed CD90, CD73, and CD105, while remaining negative for CD34 and CD45. Monolayer derived MSCs showed higher calcium deposit formation than the EB method. However, no significant differences were observed in adipogenic and chondrogenic differentiation abilities.The proliferation abilities of both methods were maintained for up to 20 passages.